Pilose antler peptide attenuates LPS-induced inflammatory reaction

doi:10.1016/j.ijbiomac.2017.11.176

International Journal of Biological Macromolecules

Volume 108, March 2018, Pages 272-276

https://doi.org/10.1016/j.ijbiomac.2017.11.176 Get rights and content

Abstract

The present study was designed to study the effects of pilose antler peptide (PAP) on primary culture of nucleus pulposus cells in intervertebral disc. We demonstrated that PAP significantly inhibited lipopolysaccharides (LPS) induced over-production of inflammatory factors including interleukin-1β (IL-1β), tumor necrosis Factor-α (TNF-α) and interleukin-6 (IL-6) in nucleus pulposus cells. PAP also attenuated increase of malondialdehyde (MDA) and decrease of superoxide dismutase (SOD) induced by LPS challenge in a concentration-dependent manner. Moreover, the expression of the protein levels of mitogen-activated protein kinase (MAPK)/nuclear transcription factor-κB(NF-κB) were increased accompanying with the LPS challenge, which were significantly reversed after PAP treatment. Our results demonstrated the ability of PAP to antagonize LPS-mediated inflammation in primary culture of nucleus pulposus in intervertebral disc, suggesting a beneficial potential for its clinical application.

Introduction

Intervertebral disc is consisted of nucleus pulposus, annulus fibrosus and cartilage endplates, which is the main structure to provide stability and flexibility to the spinal column [1], [2]. However, intervertebral disc degeneration, when comes to the condition of cell viability decrease, attenuation of type II collagen and proteoglycan synthesis, and dehydration of nucleus pulposus, has been considered as an irreversible process [3]. Also, intervertebral disc degeneration is a significant contributor to the development of low back pain and other spinal degenerative diseases [4]. Although the etiology of intervertebral disc degeneration is still obscure, a genetic component and abnormalities of inflammation and oxidative stress are strongly suggested.

Inflammation has been suggested to be closely involved with the pathophysiological process of intervertebral disc degeneration [5]. The inflammatory cytokines, such as interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), play an important role in intervertebral disc degeneration [6]. The elevated inflammatory factors are a stimulant of the inflammatory cascade. The inflammatory factors, instead of directly degrading the intervertebral disc, promote the production of inflammatory substances by the disc cells and accelerate intervertebral disc degeneration [7]. Suppression of inflammatory cytokines showed beneficial effects in preventing the intervertebral disc degeneration [8]. Restriction of interleukin-1β (IL-1β) was found to inhibit NF-κB signaling or deacetylate the transcription factors, which promotes extracellular matrix repair and defends against disc regeneration. [9]

Oxidation and reduction processes are also closely associated with the progression of intervertebral disc degeneration. Oxidative stress has been suggested to inhibit the proliferation, stimulate premature senescence and lead to a catabolic phenotype in human nucleus pulposus intervertebral disc cells [10]. The antioxidative nanofullerol was reported to prevent intervertebral disc degeneration [11], [12].

Deer antlers, namely“lu rong” in China, “nokyong” in Korea or “tokujo” in Japan, were widely used folk medicines in Asia. These soft growing tissues were applied in traditional Chinese medicine for strengthening kidney, nursing the blood treating neurosis and prolonging life. Deer antlers have been reported to exert a variety of properties, such as anti-inflammatory, anti-stress and anti-aging effects in previous research. Pilose antler peptide (PAP: MW: 7200; amino acid residue: 68) is isolated from the deer antlers and has been shown beneficial effects on chronic inflammatory and oxidative damages [13], [14], [15]. However, the role of PAP during intervertebral disc degeneration have not been investigated before. The underlying mechanisms of its actions are still obscure. The present study was designed to study the effects of PAP on LPS-induced primary culture of nucleus pulposus in intervertebral disc and explore the underlying mechanisms.

Section snippets

Reagents and kits

LPS was purchased from Sigma (St. Louis, MO, USA) and applied as an inflammatory stimulant. The drug, PAP was obtained from Changchun University of Chinese Medicine. Enzyme-linked immunosorbent assay Enzyme-linked immunosorbent assay (ELISA) kits of cytokines IL-1β, IL-6 and TNF-α were purchased from eBioscience. CO., LTD. Primary antibodies a were got from Cell Signaling Technology (Danvers, USA).

Nnucleus pulposus cells isolation and culture

The nucleus pulposus cells were extracted and isolated from the lumbar spines of Sprague Dawley

Effects of PAP on cell viability

To study the cell viability of PAP on LPS induced primary nucleus pulposus cells, we cultured nucleus pulposus cells with different concentrations of PAP (1, 4, 8, 16, 32, 64, 128 μM) for 24 h with or without LPS by CCK-8 assay. As shown in Fig. 1, PAP increased cell viability at concentrations of 4–16 μM. Thus, we chose PAP at 4, 8, 16 μM for the following experiments (Fig. 1).

Effects of PAP on inflammatory cytokines in LPS-induced primary nucleus pulposus cells

The nucleus pulposus cell cultures

Discussion

The intervertebral disc works as shock absorbers during the loading of the spine. A damaged disc is hardly to self-repair and perform functions [16]. Intervertebral disc degeneration, which is thought as a significant cause of socio-economic problems, has been suggested as a common disease closely related to the inflammation of nucleus pulposus cells [17]. Pilose antler peptide (PAP: MW: 7200; amino acid residue: 68) is isolated from the deer antlers, which has been reported to exhibit

Conflicts of interest

No potential conflicts of interest were disclosed.

Acknowledgements

This work was partly supported by National Natural Science Foundation of China (81503465, 81572168), and the Natural Science Foundation of Guangdong Province (2015A030310263).

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Pilose antler peptide attenuates LPS-induced inflammatory reaction

Abstract

Introduction

Section snippets

Reagents and kits

Nnucleus pulposus cells isolation and culture

Effects of PAP on cell viability

Discussion

Conflicts of interest

Acknowledgements

Spine

Eur. J. Pharmacol.

Biochem. Biophys. Res. Commun.

Cell Stress Chaperones

Exp. Eye Res.

Life Sci.

Am. J. Clin. Pathol.

Spine

Spine

Dissertations & Theses – Gradworks

J. Roy. Soc. Interface